The present invention relates generally to the detection of reverse transcriptase activity and, more particularly, to measuring viral reverse transcriptase quantitatively, in real time, in the context of drug screening as well as clinical testing.
Silver et al., Nucleic Acids Res. 21: 3593 (1993), disclose an approach to detect a retrovirus, in a biological sample, by PCR amplification of the cDNA product of viral reverse transcriptase (RT). By this approach, a cDNA product is synthesized only if viral RT is present, and that cDNA then is detected by a conventional technique for identifying the presence of a DNA. Techniques for this purpose include, for example, Southern blot hybridization, visualization by ethidium bromide staining, measurement of incorporated radiolabeled nucleotides, and immunoassaying for a detectable moiety bound to the amplified DNA.
The techniques of product-enhanced reverse transcriptase assay (PERT, a/k/a xe2x80x9cAmp-RTxe2x80x9d) and polymerase chain-reaction-based reverse transcriptase assay (PBRT) also have been employed to detect retroviral contamination, given their 106-fold enhanced sensitivity, relative to conventional RT assays. Thus, the FDA Center for Biologics Evaluation and Research (CBER) has recommended PERT assays for screening patients for the presence of RT, and additionally for screening viral vaccine and gene-therapy preparations for RT contamination. Also, U.S. Pat. No. 5,849,494 describes the use of these methods for assaying the presence of human immunodeficiency virus (HIV) in a biological sample. On the other hand, PERT and PBRT present significant drawbacks in a clinical setting, because these methods are laborious and cannot be effected in real time.
The application of TaqMan(copyright) technology (PE Biosystems), in conjunction with a standard PCR-RT protocol for presence of RT, is well-established for the quantitative measurement of reverse transcriptase. See Ringel et al., J. Clin. Endocrinol. and Metabolism 84: 4037 (1999). Detection of RT activity with the TaqMan(copyright) technique can be effected rapidly and relatively safely. The TaqMan(copyright) technique further represents an advance over previous methodology because of its considerably increased sensitivity over conventional RT assays, the ability to shorten measurement time to within six hours, while eliminating the need for toxic chemicals such as radioisotopes and ethidium bromide.
Lovatt et al., J. Virological Methods 82: 185 (1999), report using TaqMan(copyright) PCR technology to detect and quantify viral contamination, in unknown samples, in the laboratory setting. Although the TaqMan(copyright) technique is employed in the laboratory setting, it apparently has not been used to detect HIV reverse transcriptase. Furthermore, although reverse transcriptase is known to be inhibited by various drug agents, a method of monitoring such agents"" effectiveness by assaying RT is lacking in the clinical setting.
It is therefore an object of the present invention to provide an approach, for detecting viral RT, which is readily adapted to clinical use.
It is another object of the present invention to provide for the real-time monitoring of RT amplification products via a methodology that is particularly suited to testing agents for RT-affecting activity, e.g., in a high-throughput screening environment.
It is a further object of the present invention to assess, via the monitoring of viral RT activity, the efficacy of an anti-HIV or other anti-viral drug or putative therapeutic agent.
It is similarly an object of the invention to provide a diagnostic technique which can be performed in a clinical setting, rapidly and safely, to identify a range of pathogens of common medical concern.
It is still another object of the present invention to detect the presence of RT that is resistant, or to measure the level of resistance, to a known RT inhibitor.
In accomplishing these and other objectives, there has been provided, in accordance with one aspect of the invention, a method for measuring the presence of RT in a sample. The sample is tested for the presence of RT by contacting the sample with (i) an RNA template, such as polyadenylated RNA, (ii) a primer that is complementary to the RNA template, for example, oligo-dT, and (iii) a plurality of oligonucleotide-specific primers, at about a temperature preferably less than the deactivation temperature of the RT, which is typically in the range of about 37xc2x0 C. for HIV RT. If RT is present in the sample, then cDNA is produced and can be detected accordingly.
In another embodiment of the present invention, a method is provided for measuring the effect of a test agent on the activity of an RT in a sample. The sample, which may contain an RT, is brought into contact with an RNA template, preferably human, a complementary primer, and plurality of oligonucleotide-specific primers such that cDNA is formed only if RT is present in the sample in an amount inversely proportional to the effect of said test agent. The sample is preferably taken from a human patient and is tested in the presence of HIV RT in real time in a clinical setting.
In this vein, one skilled in the art will understand that RT in a sample, preferably from a patient, can be tested to determine whether the RT in the sample has developed resistance to a known RT inhibiting test agent. This can be accomplished by measuring the effect of a known RT inhibiting test agent on the activity of RT in a sample, comparing that activity against the activity of RT in the absence of the known RT inhibitor and/or the activity of the RT in the presence of an RT inhibitor which is known to completely inhibit the RT activity of the sample.
Other objects and advantages of the invention will become apparent by review of the detailed description of preferred embodiments.